b.**Histone modifications:**

It is the interesting and emerging mechanism that exhibits the addition of methyl groups at histones related to a gene. As DNA is structured into chromosomes in eukaryotic cells, it is tightly wrapped onto series of nucleosomes (the basic unit of chromatin), which are the octamer complexes of small core (a H3-H4 tetramer and two H2A-H2B dimers) linked by linker histone proteins (H1) [62]. These histones are involved in post-translational modifications (PTMs) which may regulate gene expressions. The gene activation and repression are determined by dynamic chromatin structure that directly depends upon these PTMs, as they will allow transformation of inactive or repressive chromatin to euchromatin, the active condition of chromatin. These modifications, like DNA methylation, are able to regulate the gene expression without any change in its DNA sequence. Hence, histone tails can be acetylated, methylated, or phosphorylated. Histones with methylated (Kme) or acetylated (Kac) lysine residues, mostly at amino terminal tails, have been identified. Generally, these modifications are correlated with either gene activation or repression. Like, on one hand, histone lysine acetylation (H3K9ac, H3K14ac and H4K5ac) is generally associated with gene activation that opens the chromatin for the binding of transcription machinery [63]. Histone acetylation is tightly controlled by the equilibrium between acetylation (HATs) and deacetylation (HDACs) enzymes that add or deletes acetyl group. On the other hand, methylation on lysine or arginine residues can be correlated with both, gene activation or gene repression, depending on the residue to be modified. For example, mono- or tri-methylation of Histone 3 at lysine 4 residue (H3K4me, H3K4me3), H3K79me2 [64] and at lysine 36 residue (H3K36me) facilitated by lysine methyl transferases (KMTs) such as SET1/7/9 are associated with gene activation [63]. Although, mono-methylation of histone 3 at lysine residue 9 (H3K9me) mediated by suppressor of variegation 3–9 homolog 1 (SUV39H1) is correlated with gene activation whilst, its trimethylation (H3K9me3) is linked with gene repression [65]. Additionally, H3K27me3 and H4K20 were associated with gene repression. Afterwards, lysine demethylases (LSD1) are there to reverse such steady modifications at H3K4 and H3K9 [66, 67] as a co-repressor or co-activator respectively. Their nomenclature has already been changed from LSD1 to lysine demethylases (KDMs) [68]. In a study in lymphocytes from type 1 diabetic patients, as compared to controls increased H3K9me2 levels were reported to be correlated with immune and inflammatory pathways associated with diabetes and its complications including DN [69]. Such histone modifications at N-terminal are two key mechanisms that may alter development and progression of diabetes and its related complications; they are noteworthy as discussed below.

**181**

via TGF-β.

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

In DN pathogenesis, expressions of a gene that are associated with DN are regulated by post-translational modifications of histone proteins, apart from DNA methylation. Smad2/3/4 (transcription factors) are activated by TGF-β and also team up with HATs and other chromatin remodeling factors. Alterations in DNA methylation and H3K9Ac at gene promoters were found to be associated with endothelial dysfunction in endothelial cells cultured in hyperglycemic conditions. Among various epigenetic mechanisms, methylation among core histone tails is considered to be the highly stable PTM that could be a key factor in the pathogenesis of various complications of diabetes. Previous studies have studied the role of histone modifications in cultured cells as well as animal model in the presence of TGF-β and high glucose environment, the two key factors in diabetes [70]. They reported an increased H3K9/14Ac at PAI-1 and p21 promoters near Smad/SP1 binding sites. Cultured rat mesangial cells (RMCs), obstructed by TGF-β antibodies, displayed increased levels of p21 and PAI-1 under hyperglycemic conditions. Also in glomeruli of diabetic animal model, increased expressions of PAI-1 and p21 were found to be linked with increased promoter H3K9/14Ac. In the model of DN, TGF-β stimulated expressions of key fibrotic genes were found to be associated with enrichment of histone active chromatin marks (H3K4me1/2/3) and reduced repressive chromatin marks (H3K9me2/3) at their promoters [71]. Collectively, TGF-β plays as an intermediator in hyperglycemia induced histone modifications of promoters of key genes in mesangial cells leading to kidney damage. In glomeruli of diabetic mice, increased chromatin active marks along with decreased repressive marks were observed at PAI-1 and receptor for AGE (RAGE) gene promoters as compared to control, which showed the regulation of histone modifications in kidney in the presence of hyperglycemia [72]. In addition, AT1R inhibitor decreased key indicators of DN and also reversed some of the epigenetic changes in diabetic mice including reduced H3K9/14Ac at PAI-1, RAGE and MCP-1 promoters in diabetic mesangial cells. In the animal models of DN, increased histone active marks (H3K4me2) and decreased repressive marks (H3K27me3) were observed to be associated with the expression of genes related to DN [73]. In the kidney of uninephrectomized db/db mice model, H3K4me2 levels were increased in association with albuminuria, glomerular filtration rate and glomerular cell proliferation, which can be reversed by MCP-1/CCL2 antagonist [74]. In diabetic kidneys, HDAC inhibitor (Trichostatin A) has been observed to block the induction of TGF-β at essential fibrotic genes, both *in vitro* and *in vivo*. This implies major role of HDACs in TGF-β facilitated kidney fibrosis and ECM accumulation [75]. In another study, treatment of renal epithelial cells with Trichostatin A (TSA) resulted in downregulated TGF-β mediated epithelial-to-mesenchymal transition (EMT) [75, 76]. Taken as a whole, these studies demonstrate the involvement of HDACs in renal injury

Histone post-translational modifications have also been studied extensively in the context of DR. Increased oxidative stress and simultaneous decreased levels of retinal superoxide dismutase (SOD2) are the key features of DR. Increased histone repressive mark (H4K20me3) along with increased NF-κB p65 in association with decreased SOD2 mRNA levels and decreased activation marks (H3K4me1/2) at SOD2 promoters were observed in retinal endothelial cells cultured in high glucose. Acetylation of core histone protein on lysine residues is thought to opens up the DNA, thereby, increased availability for binding of transcription factors. Afterwards, activated proinflammatory transcription factors, for instance NF-κB, binds to particular sequence in DNA and activates and bind coactivators (like p300) having intrinsic HAT activity to the target promoters of target gene. These coactivator molecules then, regulate the expressions of target gene owing to their HAT activity [77]. Contrary to this, recruitment of HDACs results in compact

*DOI: http://dx.doi.org/10.5772/intechopen.94642*

#### *Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

In DN pathogenesis, expressions of a gene that are associated with DN are regulated by post-translational modifications of histone proteins, apart from DNA methylation. Smad2/3/4 (transcription factors) are activated by TGF-β and also team up with HATs and other chromatin remodeling factors. Alterations in DNA methylation and H3K9Ac at gene promoters were found to be associated with endothelial dysfunction in endothelial cells cultured in hyperglycemic conditions. Among various epigenetic mechanisms, methylation among core histone tails is considered to be the highly stable PTM that could be a key factor in the pathogenesis of various complications of diabetes. Previous studies have studied the role of histone modifications in cultured cells as well as animal model in the presence of TGF-β and high glucose environment, the two key factors in diabetes [70]. They reported an increased H3K9/14Ac at PAI-1 and p21 promoters near Smad/SP1 binding sites. Cultured rat mesangial cells (RMCs), obstructed by TGF-β antibodies, displayed increased levels of p21 and PAI-1 under hyperglycemic conditions. Also in glomeruli of diabetic animal model, increased expressions of PAI-1 and p21 were found to be linked with increased promoter H3K9/14Ac. In the model of DN, TGF-β stimulated expressions of key fibrotic genes were found to be associated with enrichment of histone active chromatin marks (H3K4me1/2/3) and reduced repressive chromatin marks (H3K9me2/3) at their promoters [71]. Collectively, TGF-β plays as an intermediator in hyperglycemia induced histone modifications of promoters of key genes in mesangial cells leading to kidney damage. In glomeruli of diabetic mice, increased chromatin active marks along with decreased repressive marks were observed at PAI-1 and receptor for AGE (RAGE) gene promoters as compared to control, which showed the regulation of histone modifications in kidney in the presence of hyperglycemia [72]. In addition, AT1R inhibitor decreased key indicators of DN and also reversed some of the epigenetic changes in diabetic mice including reduced H3K9/14Ac at PAI-1, RAGE and MCP-1 promoters in diabetic mesangial cells. In the animal models of DN, increased histone active marks (H3K4me2) and decreased repressive marks (H3K27me3) were observed to be associated with the expression of genes related to DN [73]. In the kidney of uninephrectomized db/db mice model, H3K4me2 levels were increased in association with albuminuria, glomerular filtration rate and glomerular cell proliferation, which can be reversed by MCP-1/CCL2 antagonist [74]. In diabetic kidneys, HDAC inhibitor (Trichostatin A) has been observed to block the induction of TGF-β at essential fibrotic genes, both *in vitro* and *in vivo*. This implies major role of HDACs in TGF-β facilitated kidney fibrosis and ECM accumulation [75]. In another study, treatment of renal epithelial cells with Trichostatin A (TSA) resulted in downregulated TGF-β mediated epithelial-to-mesenchymal transition (EMT) [75, 76]. Taken as a whole, these studies demonstrate the involvement of HDACs in renal injury via TGF-β.

Histone post-translational modifications have also been studied extensively in the context of DR. Increased oxidative stress and simultaneous decreased levels of retinal superoxide dismutase (SOD2) are the key features of DR. Increased histone repressive mark (H4K20me3) along with increased NF-κB p65 in association with decreased SOD2 mRNA levels and decreased activation marks (H3K4me1/2) at SOD2 promoters were observed in retinal endothelial cells cultured in high glucose. Acetylation of core histone protein on lysine residues is thought to opens up the DNA, thereby, increased availability for binding of transcription factors. Afterwards, activated proinflammatory transcription factors, for instance NF-κB, binds to particular sequence in DNA and activates and bind coactivators (like p300) having intrinsic HAT activity to the target promoters of target gene. These coactivator molecules then, regulate the expressions of target gene owing to their HAT activity [77]. Contrary to this, recruitment of HDACs results in compact

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

also observed in persons with PDR [57].

b.**Histone modifications:**

progressive from non-DR stage to NPDR and eventually to PDR and were independent of hyperglycemia, dyslipidemia, diabetes duration and person's blood pressure. Binding of polymerase gamma 1 (POLG1) to mtDNA (mitochondrial DNA) also results in compromised transcriptional activity as a result of hypermethylation at promoter region of DNA polymerase gamma 1 (POLG1) in the hyperglycemic environment [42]. This study was conducted in rat model of diabetes which showed that the mitochondrial damage in retina of diabetic rats could be diminished/controlled by maintaining stable glycemic control for longer time periods or therapy that targets directly DNA methylation. However, it does not benefit DNA methylation machinery by the reversal of hyper-glycemic environment for shorter duration [59]. In people with diabetes mellitus, it has been seen that activity of Dnmt1 enzyme was elevated in retinal and its capillary cells. However, this was not observed with Dnmt-3a or Dnmt-3b [60, 61]. Similar differential DNA methylation patterns were

It is the interesting and emerging mechanism that exhibits the addition of methyl groups at histones related to a gene. As DNA is structured into chromosomes in eukaryotic cells, it is tightly wrapped onto series of nucleosomes (the basic unit of chromatin), which are the octamer complexes of small core (a H3-H4 tetramer and two H2A-H2B dimers) linked by linker histone proteins (H1) [62]. These histones are involved in post-translational modifications (PTMs) which may regulate gene expressions. The gene activation and repression are determined by dynamic chromatin structure that directly depends upon these PTMs, as they will allow transformation of inactive or repressive chromatin to euchromatin, the active condition of chromatin. These modifications, like DNA methylation, are able to regulate the gene expression without any change in its DNA sequence. Hence, histone tails can be acetylated, methylated, or phosphorylated. Histones with methylated (Kme) or acetylated (Kac) lysine residues, mostly at amino terminal tails, have been identified. Generally, these modifications are correlated with either gene activation or repression. Like, on one hand, histone lysine acetylation (H3K9ac, H3K14ac and H4K5ac) is generally associated with gene activation that opens the chromatin for the binding of transcription machinery [63]. Histone acetylation is tightly controlled by the equilibrium between acetylation (HATs) and deacetylation (HDACs) enzymes that add or deletes acetyl group. On the other hand, methylation on lysine or arginine residues can be correlated with both, gene activation or gene repression, depending on the residue to be modified. For example, mono- or tri-methylation of Histone 3 at lysine 4 residue (H3K4me, H3K4me3), H3K79me2 [64] and at lysine 36 residue (H3K36me) facilitated by lysine methyl transferases (KMTs) such as SET1/7/9 are associated with gene activation [63]. Although, mono-methylation of histone 3 at lysine residue 9 (H3K9me) mediated by suppressor of variegation 3–9 homolog 1 (SUV39H1) is correlated with gene activation whilst, its trimethylation (H3K9me3) is linked with gene repression [65]. Additionally, H3K27me3 and H4K20 were associated with gene repression. Afterwards, lysine demethylases (LSD1) are there to reverse such steady modifications at H3K4 and H3K9 [66, 67] as a co-repressor or co-activator respectively. Their nomenclature has already been changed from LSD1 to lysine demethylases (KDMs) [68]. In a study in lymphocytes from type 1 diabetic patients, as compared to controls increased H3K9me2 levels were reported to be correlated with immune and inflammatory pathways associated with diabetes and its complications including DN [69]. Such histone modifications at N-terminal are two key mechanisms that may alter development and progression of diabetes and its related complications; they are noteworthy as discussed below.

**180**

chromatin, coiled DNA and less accessibility for binding of transcription factors to DNA, thereby decreased expression of target gene. Hence, the balance between acetylation and deacetylation of histones regulates the transcription of the gene. Increased HDACs and decreased HATs along with decreased global histone acetylation activities were also found in diabetic retinal cells in the models of diabetic retinopathy [78]. However, reversal of hyperglycemic conditions did not able to restore changes in histone activities. This is in contrast to a study in diabetes where activation of histone acetylation was observed in retinal cells [79]. Pro-apoptotic enzyme, MMP-9, is also observed to be associated with epigenetic alterations in DR [80, 81]. Lysine of histone 3 was reported to be methylated by SUV39H1 resulted in H3k9me3 [82]. Another methyl transferase gene i.e., SUV39H2 is involved in the onset of disease, when methylates histone H3K9 results in the inception of DR [83]. Moreover under hyperglycemic conditions, recruitment of Set7 (HMT) at promoter region of NF-KB p65 unit was linked with its enhanced transcription [44]. Western blotting and mass spectrometry studies in diabetic rat model also confirmed the acetylation of several lysine residues on histones due to hyperglycemia leading to increased expressions of proinflammatory proteins in retina and associated with DR [79].

Oxidative stress also plays a central role in diabetic complications and has been shown to control histone acetylation or deacetylation in diabetic conditions. High blood glucose is known to increase ROS production, which further activates important pathways that are required for the development of DR [84]. ROS is observed to inhibit acetylation of histones by increasing HDAC activity and decreasing HAT activity [85]. Hence, it was believed that there is involvement of ROS in regulating acetylation and deacetylation. Usually, oxidative stress was found to be increased in retina and capillary cells [86]. Thus, it is possible that diabetes via increased ROS production may regulates histone acetylation and deacetylation in retina. Ischemia and hypoxia are also known to promote the process of histone deacetylation [87] and hypoxia in diabetes is the leading cause for neovascularization in retina [88] which indicates the role of retinal hypoxia in diabetic retinopathy via stimulating retinal histone deacetylases. Thus, in hyperglycemia, epigenetic alterations may be involved at a larger level in modulating the expressions of various important genes in pathogenesis of DR.

Various researches on histone protein alterations may suggests that chromatin state is likely to be affected by multiple histone code modifications and hence, screening of various histone alterations at key genes promoters and/or bodies related to DN is crucial. The role of DNA methylation, histone code modifications and changes in epigenetic marks in response to various therapies is not well studied and would be of great concern to see whether these modifications could be altered in response to therapy. In future, more epigenome studies are required to elucidate the mechanisms of pathogenesis of DN that could help in developing better treatment strategies for people suffering from this devastating complication.

#### c.**Micro RNAs (miRNA):**

Whole transcriptome studies (RNA-sequencing) have uncovered that majority of the transcribed genome (into RNA) is non-coding part, apart from the coding mRNA [89]. Non-coding RNA refers to the RNA that does not code for any protein. These non-coding RNAs are also a part of epigenetic mechanisms that are of immense interest in the context of diabetic complications as they are observed to repress the expressions of target genes via regulating transcription and post-transcription mechanisms. Non-coding RNAs includes small non-coding RNAs (miRNAs approx. 20-22 bp long), circular RNAs (circRNAs) as well as long

**183**

diabetes complications.

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

non-coding RNAs (lncRNAs approx. 200 bp long). They are reported to control the expressions of important genes associated with diabetic complications. In contrast to miRNAs, few studies have observed the role of lncRNAs in DN [90, 91]. miRNAs are usually single stranded RNA of approximately 20–25 nucleotides long. They are well-known non-coding RNAs that involved in post-transcriptional regulation by means of either suppression of translation or degradation of mRNA transcript by binding 3' UTR of target sequences [92, 93]. LncRNAs, instead, are usually longer (>200 bp) than miRNAs (20-22 bp). They function as scaffolds [94] and may regulate miRNA due to their antisense activity [95] and have tissue specific expressions [96]. Similar to mRNA, lncRNAs are formed due to transcription in the presence of RNA polymerase II and undergo splicing, although they are slightly polyadenylated [96]. LncRNAs also participate in modifications of epigenetic marks as they harbor histone methylation marks at H3k4 and H3K36 [97]. They are also reported to be involved in the development and progression of diabetic microvascular complications [98–101]. Recently circular RNAs, the next level of epigenetic regulation, are holding our interest in addition to lncRNAs as they are generated from mRNA via its back-splicing and later on both 5′ and 3′ spliced ends ligated together to form a circular structure. They regulate miRNAs, thereby regulating the expressions of miRNAs targetted genes. They also act as sponge for various miRNAs. Several circRNAs are observed to stimulate the pathogenesis of diabetes-related microvascular

On the other hand miRNAs, at first, were portrayed in *C. elegans*, a nematode, during early 1990s. Over 1000 miRNAs in human genome have been identified; lin-4 was the first described miRNA [104]. Various miRNAs are found in humans, algae, plants, animals and viruses [105]. miRNAs, unlike other small RNAs, are derived from the transcripts that themselves can rapidly fold back to form a hairpin-like structure. RNA polymerase II transcribed miRNA as primary transcript (pri-miRNA) in nucleus, where they are later spliced into precursor miRNAs (pre-miRNA) [106, 107] by the action of endonuclease complex. Exportin-5, a protein transport pre-miRNA into the cytoplasm from nucleus where they are further processed to mature miRNA duplex (~ 22 nucleotides) by the action of ribonucleases [107]. One strand of mature miRNAs is selected and loaded on RNA induced silencing complex (RISC) and other stand undergoes the process of degradation [106, 108]. This complex binds to their complementary sequence on mRNA for post-transcriptional suppression. Initially, lin-4 RNA was observed to have complementarity with conserved sites in mRNA of lin-14 [109] within untranslated (3'-UTR) site. But how to find their targets was the primary question in initial times. Algorithm tool, at first, identifies the perfect Watson-Crick pairing to 2–8 nucleotides of miRNAs starting from 5'region [110]. This 7 seven nucleotide sequence (at 5′-end) was termed as 'miRNA seed'. This finding was clearly in agreement with the earlier study which showed that 5′ end is the most conserved region in metazoan miRNAs [111]. Afterwards extending seed match with adding more base pairs to the miRNA continues in both directions, but stopping at discrepancies [110]. Therefore, the silencing effect of target gene by miRNA is via binding of seed sequence at miRNA with the complementary sequence at mRNA in 3'-UTR. miRNAs based therapies would have a better lead in that they can target multiple genes of a particular pathway or process [112]. Because, one miRNA can supress expression of many genes and subsequently one gene can also be targetted by more than one miRNAs. Another advantage is that these miRNAs can cross blood-retina barrier so as to get into the target tissue, which is the foremost obligation with this therapy. In past years, several studies have linked miRNAs with diabetic complications. Henceforth we have, now described the role of miRNAs in the pathogenesis of

*DOI: http://dx.doi.org/10.5772/intechopen.94642*

complications [102, 103].

#### *Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

non-coding RNAs (lncRNAs approx. 200 bp long). They are reported to control the expressions of important genes associated with diabetic complications. In contrast to miRNAs, few studies have observed the role of lncRNAs in DN [90, 91]. miRNAs are usually single stranded RNA of approximately 20–25 nucleotides long. They are well-known non-coding RNAs that involved in post-transcriptional regulation by means of either suppression of translation or degradation of mRNA transcript by binding 3' UTR of target sequences [92, 93]. LncRNAs, instead, are usually longer (>200 bp) than miRNAs (20-22 bp). They function as scaffolds [94] and may regulate miRNA due to their antisense activity [95] and have tissue specific expressions [96]. Similar to mRNA, lncRNAs are formed due to transcription in the presence of RNA polymerase II and undergo splicing, although they are slightly polyadenylated [96]. LncRNAs also participate in modifications of epigenetic marks as they harbor histone methylation marks at H3k4 and H3K36 [97]. They are also reported to be involved in the development and progression of diabetic microvascular complications [98–101]. Recently circular RNAs, the next level of epigenetic regulation, are holding our interest in addition to lncRNAs as they are generated from mRNA via its back-splicing and later on both 5′ and 3′ spliced ends ligated together to form a circular structure. They regulate miRNAs, thereby regulating the expressions of miRNAs targetted genes. They also act as sponge for various miRNAs. Several circRNAs are observed to stimulate the pathogenesis of diabetes-related microvascular complications [102, 103].

On the other hand miRNAs, at first, were portrayed in *C. elegans*, a nematode, during early 1990s. Over 1000 miRNAs in human genome have been identified; lin-4 was the first described miRNA [104]. Various miRNAs are found in humans, algae, plants, animals and viruses [105]. miRNAs, unlike other small RNAs, are derived from the transcripts that themselves can rapidly fold back to form a hairpin-like structure. RNA polymerase II transcribed miRNA as primary transcript (pri-miRNA) in nucleus, where they are later spliced into precursor miRNAs (pre-miRNA) [106, 107] by the action of endonuclease complex. Exportin-5, a protein transport pre-miRNA into the cytoplasm from nucleus where they are further processed to mature miRNA duplex (~ 22 nucleotides) by the action of ribonucleases [107]. One strand of mature miRNAs is selected and loaded on RNA induced silencing complex (RISC) and other stand undergoes the process of degradation [106, 108]. This complex binds to their complementary sequence on mRNA for post-transcriptional suppression. Initially, lin-4 RNA was observed to have complementarity with conserved sites in mRNA of lin-14 [109] within untranslated (3'-UTR) site. But how to find their targets was the primary question in initial times. Algorithm tool, at first, identifies the perfect Watson-Crick pairing to 2–8 nucleotides of miRNAs starting from 5'region [110]. This 7 seven nucleotide sequence (at 5′-end) was termed as 'miRNA seed'. This finding was clearly in agreement with the earlier study which showed that 5′ end is the most conserved region in metazoan miRNAs [111]. Afterwards extending seed match with adding more base pairs to the miRNA continues in both directions, but stopping at discrepancies [110]. Therefore, the silencing effect of target gene by miRNA is via binding of seed sequence at miRNA with the complementary sequence at mRNA in 3'-UTR. miRNAs based therapies would have a better lead in that they can target multiple genes of a particular pathway or process [112]. Because, one miRNA can supress expression of many genes and subsequently one gene can also be targetted by more than one miRNAs. Another advantage is that these miRNAs can cross blood-retina barrier so as to get into the target tissue, which is the foremost obligation with this therapy. In past years, several studies have linked miRNAs with diabetic complications. Henceforth we have, now described the role of miRNAs in the pathogenesis of diabetes complications.

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

chromatin, coiled DNA and less accessibility for binding of transcription factors to DNA, thereby decreased expression of target gene. Hence, the balance between acetylation and deacetylation of histones regulates the transcription of the gene. Increased HDACs and decreased HATs along with decreased global histone acetylation activities were also found in diabetic retinal cells in the models of diabetic retinopathy [78]. However, reversal of hyperglycemic conditions did not able to restore changes in histone activities. This is in contrast to a study in diabetes where activation of histone acetylation was observed in retinal cells [79]. Pro-apoptotic enzyme, MMP-9, is also observed to be associated with epigenetic alterations in DR [80, 81]. Lysine of histone 3 was reported to be methylated by SUV39H1 resulted in H3k9me3 [82]. Another methyl transferase gene i.e., SUV39H2 is involved in the onset of disease, when methylates histone H3K9 results in the inception of DR [83]. Moreover under hyperglycemic conditions, recruitment of Set7 (HMT) at promoter region of NF-KB p65 unit was linked with its enhanced transcription [44]. Western blotting and mass spectrometry studies in diabetic rat model also confirmed the acetylation of several lysine residues on histones due to hyperglycemia leading to increased expressions of proinflammatory proteins in retina and associated

Oxidative stress also plays a central role in diabetic complications and has been shown to control histone acetylation or deacetylation in diabetic conditions. High blood glucose is known to increase ROS production, which further activates important pathways that are required for the development of DR [84]. ROS is observed to inhibit acetylation of histones by increasing HDAC activity and decreasing HAT activity [85]. Hence, it was believed that there is involvement of ROS in regulating acetylation and deacetylation. Usually, oxidative stress was found to be increased in retina and capillary cells [86]. Thus, it is possible that diabetes via increased ROS production may regulates histone acetylation and deacetylation in retina. Ischemia and hypoxia are also known to promote the process of histone deacetylation [87] and hypoxia in diabetes is the leading cause for neovascularization in retina [88] which indicates the role of retinal hypoxia in diabetic retinopathy via stimulating retinal histone deacetylases. Thus, in hyperglycemia, epigenetic alterations may be involved at a larger level in modulating the expressions of various important genes

Various researches on histone protein alterations may suggests that chromatin state is likely to be affected by multiple histone code modifications and hence, screening of various histone alterations at key genes promoters and/or bodies related to DN is crucial. The role of DNA methylation, histone code modifications and changes in epigenetic marks in response to various therapies is not well studied and would be of great concern to see whether these modifications could be altered in response to therapy. In future, more epigenome studies are required to elucidate the mechanisms of pathogenesis of DN that could help in developing better treat-

Whole transcriptome studies (RNA-sequencing) have uncovered that majority of the transcribed genome (into RNA) is non-coding part, apart from the coding mRNA [89]. Non-coding RNA refers to the RNA that does not code for any protein. These non-coding RNAs are also a part of epigenetic mechanisms that are of immense interest in the context of diabetic complications as they are observed to repress the expressions of target genes via regulating transcription and post-transcription mechanisms. Non-coding RNAs includes small non-coding RNAs (miRNAs approx. 20-22 bp long), circular RNAs (circRNAs) as well as long

ment strategies for people suffering from this devastating complication.

**182**

with DR [79].

in pathogenesis of DR.

c.**Micro RNAs (miRNA):**

Several miRNAs including miR-29, miR-192, miR-194, miR-200b/c, miR-204, miR-215, miR-216a, miR-217, miR-377 etc. have been found to be associated with DN. Characteristics of DN includes fibrosis, accumulation of extracellular matrix (ECM), podocyte dysfunction and proteinuria [113, 114]. TGF-β has been implicated in the pathogenesis of DN and is found to be upregulated during the progression of DN, which in turn, induce fibrotic events, kidney deterioration and dysfunction [114]. TGF-β has shown to upregulate several miRNAs including miR-192, miR-216a, miR-217 in mesangial cells as well as in kidneys of diabetic mouse models as compared to control group [115–117]. ZEB2, a translation repressor that supress fibrotic gene collagen type 1 Alpha 2 (*Col1a2*), was observed to get suppressed by miR-192, thus, resulted in an increased expression of *Col1a2* gene and contribute to matrix accumulation and kidney fibrosis in DN model [115]. In diabetic mice, increased expressions of p53, TGF-β and miR-192 was reported in renal cortex and was found to be associated with augmented fibrosis and glomerular expansion as compared to control. Moreover, knockout of miR-192 gene resulted in decreased markers of DN. However, conflicting reports to these results are also described. One of such reports observed that TGF-β decreased the expression of miR-192 in cultured proximal tubule cells and concluded that decreased miR-192 levels are associated with increased fibrogenesis in PTCs [118]. Another study also showed that kidney fibrosis was associated with the loss of miR-192 [119]. These contradictory studies showed that the interconnection between DN and miR-192 is much more complicated than it seems. Also, a decreased expression of miR-21 was found in DN and albuminuria was decreased in diabetic mice due to ectopic expression of miR-21 [120]. miR-377 expression was found to be upregulated in DN [121]. It actually alters the levels of MnSOD and PAK1, which in turn, resulted in augmented fibronectin expression in mesangial cells in streptozotocin (STZ) induced diabetic model, thus contributing to DN progression indirectly. TGF-β induced miR-216a expression has been shown to increased collagen (*Col1a2*) expression [116] and subsequently participates in the fibrogenesis in proximal tubular cells (PTCs) [122]. Another important contributor to DN is VEGF and treatment with anti-VEGF showed to improve kidney functions in diabetic animal model [123]. Earlier miRNA-93 was considered as 'signature miRNA' in both *in vivo* as well as *in vitro* hyperglycemic environment [124]. Long et al. also demonstrated that increased expression of miR-93 resulted in reduced high glucose-stimulated VEGF-A levels via downregulation of the host *MCM7* gene promoter.

Earlier studies have also reported the role of miRNAs in diabetic retinopathy. Neovascularization is the hallmark of DR and several studies have confirmed the importance of miRNAs in neovascularization regulation in retina [125]. Microarray studies recognized increased (miR-146, miR-106a, miR-181, miR-199a, miR-214, miR-424 and miR-451) as well as decreased expressions of various miRNAs (miR-31, miR-150, miR-184) in model of ischemic retinopathy [126]. In retina and retinal endothelial cells (RECs), increased miRNAs corresponding to NF-κB, p53 and VEGF were identified reflecting pathological changes of early DR by means of functional analysis, thus, revealing the role of miRNA in pathogenesis of DR [127]. In diabetes, downregulated miR-200b was detected in retina of diabetic rat model with simultaneous elevated levels of VEGF mRNA and protein. In addition, *in vitro* miR-200b antagonist transfection resulted in elevated VEGF expression [128]. This demonstrates VEGF to be the direct target of miR-200b. During early stage of diabetes, miR-29 shown to be anti-apoptotic for retinal ganglion cells (RGCs) and inner nuclear layer (INL) cells through pro-apoptotic RNA dependent (PKR) signaling pathway [129].

Therefore, this chapter has enlightened the role and contribution of epigenetic mechanisms in the pathogenesis of two major diabetic vascular complications i.e.,

**185**

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

DN and DR. Together all, it indicates the important connection of miRNAs with microvascular complications of diabetes; hence, it would be worth to explore the role of these alterations in the pathophysiology of DN as well as DR. As reviewed in this chapter, methylation in DNA, histone tail alterations and variable expressions of miRNAs are found to be altered in hyperglycemic environment either upregulated or downregulated affecting directly or indirectly. Current treatment for DN and DR is not able to stop the progression of these devastating complications, henceforth, focusing treatment approaches via targeting epigenetic alterations alone or in combination with conventional therapy could provide a new approach to combat or retard the progression of these diabetic complications. However, the fact that a particular miRNA can have multiple targets made it difficult and challenging with few limitations, still it will increase our understanding about the disease

**4. Targeting diabetic complications via targeting epigenetic marks**

ing NF-κB expression [136], SIRT1 has been shown to possess NAD+

protein deacetylase activity [137]. In glomerular mesangial cells, SIRT 1 induces antioxidant genes and simultaneously downregulates TGF-β1 and the expression of AGEs-induced fibronectin [138]. In diabetic mice glomeruli, BF175, a SIRT1 agonist, ameliorates hyperglycemia-induced podocyte loss, proving the protective role of SIRT1 against diabetes-induced kidney damage [139]. Recently, angiotensin II (Ang II) of RAAS has been reported to induce the expressions of few non-coding RNAs including miRNAs [140] and lncRNAs [141] as well. Enhancers, the elements that affect transcription of genes and are associated with specific histone modifications [142], when blocked by JQ1, a Bromodomain (an epigenetic reader) inhibitor, also obstructs enhancer functions along with attenuation of Ang II-mediated hypertension and inflammation *in vivo* in vascular smooth muscle cells (VSMCs) [143], hence, strongly supporting the importance of targeting enhancers in Ang II-mediated actions for treating vascular complications. This, in turn, could reveal evidence directing new therapeutic interventions for treatment of diabetic vascular complications. In addition, the modified inhibitor of miR-192 i.e., Locked nucleic acid (LNA) not only downregulates key fibrotic markers of kidney damage but also shown to reduce proteinuria in diabetic mice [144], favoring miRNAs based therapeutic interventions for DN. Several studies have also reported the amelioration of kidney-injury parameters via targeting miR-21 [145–147] implying that its inhibition could be a promising therapeutic intervention in DN. Recently with the use of latest and advanced approach of genome editing i.e., CRISPR-Cas9, locus-specific changes in epigenetic alterations could be generated owing to the fusion of Cas9 proteins with various DNMTs or TETs or histone modification proteins [148–150],

Heritable epigenetic alterations are the results of interactions between environmental (momentary) and genetic (long-standing) components and thus, may play a decisive role in the pathophysiology of diabetic complications. They are able to alter the gene expression, thereby, gene function, the underline mechanism in the pathogenesis of vascular complications of diabetes. Reversible attribute of epigenetic marks provides immense opportunity of developing restorative interventions for treating patients with these complications. Till date, some of the drugs targeting epigenetic marks are already being clinically used for cancer therapy including HDAC inhibitors [130] and DNA methylation inhibitors [131, 132]. However, preclinical studies targeting histone as well as DNA methylation are still in progress [133–135]. Metformin, the current line of drug for treating hyperglycemia, upregulates sirtuin 1 (SIRT1) expression along with downregulat-


*DOI: http://dx.doi.org/10.5772/intechopen.94642*

pathophysiology.

#### *Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

DN and DR. Together all, it indicates the important connection of miRNAs with microvascular complications of diabetes; hence, it would be worth to explore the role of these alterations in the pathophysiology of DN as well as DR. As reviewed in this chapter, methylation in DNA, histone tail alterations and variable expressions of miRNAs are found to be altered in hyperglycemic environment either upregulated or downregulated affecting directly or indirectly. Current treatment for DN and DR is not able to stop the progression of these devastating complications, henceforth, focusing treatment approaches via targeting epigenetic alterations alone or in combination with conventional therapy could provide a new approach to combat or retard the progression of these diabetic complications. However, the fact that a particular miRNA can have multiple targets made it difficult and challenging with few limitations, still it will increase our understanding about the disease pathophysiology.
